We have used genetic, biochemical, and transcriptomic analysis techniques to interrogate Variovorax paradoxus EPS behavior on surfaces. We have observed that surfactant production is crucial to motility, is regulated by environmental conditions, and is responsive to the activation of a two component system, shkRS. We have also identified the locus responsible at least in part for surfactant biosynthesis, Varpa_4519, and identified the surfactant molecule by imaging mass spectrometry. Finally, we have demonstrated that V. paradoxus has anti-staphylococcal activity when co-cultured on agar plates, and that this activity is related to mucoid colony phenotypes and surface motility. In this proposal we will investigate the regulon controlled by the ShkRS two component system, to determine its role in controlling the transition between sessile and motile surface associated behaviors. We will also examine the structural and functional characteristics of the novel biosurfactant variowettin, along with the regulatory network that controls its synthesis. Finally, we will investigate the anti- staphylococcal activity that we have observed to determine what secondary metabolites are involved, with special emphasis on the potential for variowettin as a possible antimicrobial. We will expand our investigation of the anti-staphylococcal activity to tes for biofilm invasion or disruption, to determine if V. paradoxus EPS can also kill or inhibit pathogenic and enterotoxigenic strains of S. aureus, and to determine if the activity is broadly inhibitory to other microorganisms. These experiments will help determine if V. paradoxus EPS has potential as a probiotic organism, or if it's secondary metabolites have potential as therapeutics.